Method of treating bladder and lower urinary tract syndromes

ABSTRACT

The present invention relates to bladder and lower urinary tract syndromes, particularly, irrative symptoms, and to a method of treating same using α 1d -adrenergic receptor (α 1d dAR) antagonists. The invention further relates to a method of screening compounds for their ability to serve as α 1d AR selective antagonists.

This application is a division of application Ser. No. 10/268,969, filedOct. 11, 2002, which is a continuation of application Ser. No.09/306,013, filed May 6, 1999, now abandoned, which claims priority fromU.S. Provisional Application No. 60/084,479, filed May 6, 1998, theentire contents of these applications being incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to bladder and lower urinary tractsyndromes, particularly, irritative symptoms, and to a method oftreating same using α_(1d)-adrenergic receptor (α_(1d)AR) antagonists.The invention further relates to a method of screening compounds fortheir ability to serve as α_(1d)AR antagonists.

BACKGROUND

Lower urinary tract symptoms (LUTS) resulting from bladder outletobstruction (BOO) remains one of the most commonly encountered disordersin urology, and can be secondary to fixed anatomical and/or functionalcauses (Steers et al, Voiding dysfunction: diagnosis, classification,and management, in Adult and Pediatric Urology; Third Edition, J. Y.Gillenwater, et al., Editors. 1996, Mosby-Year Book, Inc.: St. Louis. p.1220-1325.). Causes of BOO include prostatic enlargement (benign ormalignant), bladder neck contracture, urethral stricture, and meatalstricture (Steers et al, Voiding dysfunction: diagnosis, classification,and management, in Adult and Pediatric Urology; Third Edition, J. Y.Gillenwater, et al., Editors. 1996, Mosby-Year Book, Inc.: St. Louis. p.1220-1325.). Symptoms associated with BOO typically fall intoobstructive or irritative categories; obstructive symptoms includehesitancy, poor stream, prolonged urination, and feelings of incompleteemptying, while irritative symptoms consist of frequency, urgency,nocturia, and unstable bladder contractions. The bladder is functionallyand anatomically divided into the detrusor (body and ventral base) andtrigone (dorsal portion of base extending between the ureteral orificesand the bladder neck) (Zderic et al. Voiding function: relevant anatomy,physiology, pharmacology, and molecular aspects, in Adult and PediatricUrology; Third Edition, J. Y. Gillenwater, et al., Editors. 1996,Mosby-Year Book, Inc.: St. Louis. p. 1159-1219), with distincthistology, histochemistry, and pharmacology. In contrast, the prostateand trigone have similar vascular supply, innervation, and receptorexpression (Gosling et al, Detrusor morphology in relation to bladderoutflow obstruction and instability. in Benign Prostatic Hypertrophy, F.Hinman, Editor. 1983, Springer-Verlag: Berlin. p. 666-71).

The physiology of LUTS secondary to benign prostatic hypertrophy (BPH)has two components: (1) a static component related to the increase inprostatic cellular mass and (2) a dynamic component related tovariations in prostatic smooth muscle tone (Caine et al, Brit. J. Urol.47:193-202 (1975)). Histologically BPH is characterized by glandular(epithelial) and stromal (fibromuscular) hyperplasia, with the latterbeing the dominant factor in the pathogenesis of clinically significantBPH (Shapiro et al, J. Urol. 147: 1293-1297 (1992)). Therefore muchattention has focused on the role of the sympathetic nervous system andα₁-adrenergic receptors (α₁ARs) in the dynamic component of BOO, leadingto clinical studies of α₁AR antagonists as agents to relieve outletobstruction. These studies have found that α₁AR antagonists relaxprostatic smooth muscle, relieving obstructive symptoms (Chapple, Brit.J. Urol. 1:47-55 (1995), Caine, Urol. Clin. N. Am. 17:641-649 (1990),Kawabe and Niijima, Urol. Int. 42:280-284 (1987), Lepor et al, J. Urol.148:1467-1474 (1992), Reuther and Aagaard. Urol. Int. 39:312-313 (1984),Matvus and Horvath. Med. Res. Rev. 17:523-535 (1997)). In addition, α₁ARantagonists have been found to relieve the irritative bladder symptomsin men (most often associated with BPH) and women (Matyus and Horvath,Med. Res. Rev. 17:523-535 (1997), Serels and Stein, Neurourol. Urodyn.17:31-36 (1998)). While it is logical to assume that elimination of BOOwould relieve irritative symptoms, a number of recent studies suggestthat the relationship between bladder irritability and outletobstruction is not straightforward (Caine, Urol. Clin. N. Am. 17:641-649(1990), Chapple and Smith, Brit. J. Urol. 73:117-123 (1994), Steers andDe, J. Urol. 140:864-71 (1988), Steers et al, Am. J. Physiol. 266:R20(1994)).

α₁ARs are members of the larger family of G protein-coupled adrenergicreceptors which mediate actions of the endogenous catecholaminesnorepinephrine (NE) and epinephrine, resulting in smooth musclecontraction. cDNAs encoding three distinct α₁AR subtypes (α_(1a),α_(1b), α_(1d)) have been cloned, expressed in cells, and resultantprotein characterized pharmacologically (Schwinn et al, J. Pharmacol.Exper. Ther. 272:134-142 (1995), Hieble et al, Pharmacol. Rev. 47:267-70(1995)). α_(1a)ARs predominate in prostate and bladder trigone (Price etal, J. Urol. 150:546-551 (1993)), and have been shown to be functionallyimportant in mediating prostate smooth muscle contraction (Forray et al,Mol. Pharmacol. 45:703-708 (1994), Lepor et al., J. Pharmacol. Exper.Ther. 270:722-727 (1994)). In addition to the three cloned α₁AR subtypeswhich have high affinity for the antagonist prazosin, a fourth type ofα₁AR with low affinity for prazosin (α_(1L)) has been postulated(Muramatsu et al, Brit. J. Urol. 74:572-578 (1994)). In spite of initialevidence suggesting a role for the α_(1L)AR in human prostate smoothmuscle contraction (Ford et al, Mol. Pharmacol. 49:209-215 (1996)), morerecent data suggests RS17053 (the compound used in these studies)detects a low affinity state of the α_(1a)AR in tissues rather than adistinct α_(1L)AR (Ford et al, Br. J. Pharmacol. 121:1127-1135 (1997)).Since non-selective α₁AR antagonists currently used to treat BPH haveundesirable side-effects including light headedness, dizziness, andasthenia (Carruthers, Drug Safety 11: 12-20 (1994)), many investigatorshave suggested that α_(1a)AR subtype selective antagonists might bebeneficial in improving BPH-related symptoms via relieving BOO (Matyusand Horvath, Med. Res. Rev. 17:523-535 (1997), Hieble and Ruffolo, Jr.,Exp. Opin. Invest. Drugs 6:367-387 (1997)). However, this approach doesnot take into account that irritative symptoms may persist in spite ofrelief of outlet obstruction (Hieble and Ruffolo, Jr.. Exp. Opin.Invest. Drugs 6:367-387 (1997)).

Very little information exists regarding the role of α₁ARs in humandetrusor. One of the few studies addressing this issue suggests humanbladder (dome) contains only α_(1a)ARs (Walden et al, J. Urol.157:1032-1038 (1997)). However, since irritative bladder symptomspersist in some patients despite relief of BOO, nonselective α₁ARantagonists may relieve the irritative effects of BPH through directeffects on bladder detrusor or other sites involved in micturation. Thepresent invention results from the realization that human detrusorexpresses two α₁AR subtypes (α_(1d)>α_(1a)). This realization makespossible the identification of α₁AR subtype selective antagonists thatcan be used to treat irritative symptoms.

SUMMARY OF THE INVENTION

The present invention relates generally to bladder and lower urinarvtract syndromes and, more particularly, to a method of identifyingα_(1d)AR antagonists that can be used to treat irritative symptoms. Theinvention also relates to a method of treating irritative symptoms usingsuch agents.

Objects and advantages of the invention will be apparent from thedetailed description that follows.

BRIEF DESCRIPTION OF THE DRAWRNGS

FIGS. 1A-1C. Schematic of the location of α₁AR subtype probes.Highlighted in bold are regions of α_(1a) (FIG. 1A), α_(1b) (FIG. 1B),and α_(1d) (FIG. 1C) ARs encoded by probes used in RNase protectionassays.

FIG. 2. Representative saturation binding isotherm generated usingincreasing concentrations of the α₁AR radiolabeled antagonist [¹²⁵I]HEATin human detrusor membranes. Kd is 130±1.09 pM (n=5), similar to thatreported for cells stably expressing each cloned human α₁AR subtype(Schwinn et al, J Pharmacol. Exper. Ther. 272:1 34-142 (1995)—α_(1a/d)ARof the reference refers to the α_(1d)AR subtype described herein sincethe α₁AR nomenclature used here is the IUPHAR nomenclature (Hieble etal, Phar. Rev. 97:267 (1995)).

FIG. 3. RiNase protection assavs examining α₁AR subtype expression indetrusor were performed in all patients (n=13). A representative RNaseprotection assay showing results from five patients is shown. In thisexperiment, radiolabeled probe for each α₁AR subtype is shown at the farleft along with (from left to right) protected fragments resulting fromtotal RNA extracted from rat-1 fibroblast cells stably expressing eachcloned human α₁AR subtype (20 mg; positive probe control), yeast tRNA(20 mg; negative control); and total RNA isolated from human detrusor(20 mg) from five patients (lanes 1-5). Gel exposure times are 24 hrsfor probe and positive control lanes and 72 hrs for tRNA and humandetrusor samples. Although the α_(1d)AR subtype mRNA band is strongerthan the α_(1a)AR protected fragment, the α_(1a)AR probe contains 73%more radiolabeled αUTP compared with the α_(1d); hence, afternormalization for radioactive label incorporation, two-fold predominanceof the α_(1d)AR subtype in human detrusor is apparent.

FIGS. 4A and 4B. Results from RT-PCR experiments on human detrusor (FIG.4A) and rat whole bladder (FIG. 4B) RNA. α₁AR subtype specific cDNA inplasmid vectors served as positive controls.

FIG. 5. α₁AR subtype expression in human detrusor was determined usingcompetition analysis with the α_(1d)AR-subtype selective ligand BMY7378.Results from a representative curve are shown demonstrating a two-sitefit with high affinity Ki corresponding to the α_(1d)AR.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the recognition of α_(1d)AR as theα₁AR subtype responsible for irritative symptoms associated with bladderand lower urinary tract diseases. The invention provides, in oneembodiment, a method of selecting α_(1d)AR antagonists and, in a furtherembodiment, a method of treating irritative symptoms using α_(1d)ARantagonists. (The nomenclature used herein is the new nomenclatureprovided in Hieble et al, Phar. Rev. 97:267 (1995)).

The method of treatment to which the invention relates comprisesadministering to a patient suffering irritative symptoms an amount of anα_(1d)AR antagonist sufficient to relieve such symptoms. In accordancewith the invention, irritative symptoms include excessive frequency ofurination, urgency of urination, nocturia and unstable bladdercontractions. Patients amenable to treatment include men and women,children and adults. In males, a preferred antagonist is both anα_(1a)AR and an α_(1d)AR antagonist. In females, preferred antagonistsare α_(1d)AR specific antagonists. The amount of the antagonist to beadministered and the treatment regimen will vary with the antagonist,the patient and the effect sought. Optimum doses and regimens, however,can be readily determined by one skilled in the relevant art.

The present invention also relates to a method of screening compoundsfor their ability to bind primarily to α_(1d)AR and thereby to function,potentially, as α_(1d)AR antagonists Preferred α_(1d)AR selectiveantagonists show at least a two fold selectivity for α_(1d)AR relativeto α_(1a)AR or α_(1b)AR. Binding assays of this embodiment inventioninclude cell-free assays in which α_(1d)AR. or portion thereof (e.g.relevant transmembrane portion—see, generally, Hwa et al, J. Biol. Chem.271:7956 (1996)). is incubated with a test compound (proteinaceous ornon-proteinaceous) which, advantageously, bears a detectable label(e.g.. a radioactive or fluorescent label). Preparations of membranesthat bear α_(1d)AR can be used in this assay, including commerciallyavailable preparations (e.g. the NEN multireceptor kit (NET 1034)).Following incubation, the α_(1d)AR, or portion thereof, free or bound totest compound, can be separated from unbound test compound using any ofa variety of techniques (for example, the α_(1d)AR (or portion thereof)(e.g., associated with a membrane) can be bound to a solid support(e.g., a plate or a column) and washed free of unbound test compound).The amount of test compound bound to α_(1d)AR, or portion thereof, isthen determined using a technique appropriate for detecting the labelused (e.g., liquid scintillation counting in the case of a radiolabelledtest compound). (See Schwinn et al, J. Pharm. Exp. Ther. 272:134(1995).)

Binding assays of this embodiment can also take the form of cell-freecompetition binding assays. Such assays can be conducted as described inthe Examples that follow (see particularly Example 2 (the test compoundbeing substituted for BMY 7378 )—see also Schwinn et al, J. Pharm. Exp.Ther. 272:134 (1995)). Alternatively, α_(1d)AR, or portion thereof, canbe incubated with a compound known to interact, specifically, withα_(1d)AR (e.g., BMY7378), which compound, advantageously, bears adetectable label (e.g., a radioactive or fluorescent label). A testcompound (proteinaceous or non-proteinaceous) is added to the reactionand assayed for its ability to compete with the known (labeled) compoundfor binding to α_(1d)AR, or portion thereof. Free known (labeled)compound can be separated from bound known compound, and the amount ofbound known compound determined to assess the ability of the testcompound to compete. This assay can be formatted so as to facilitatescreening of large numbers of test compounds by linking the α_(1d)AR, orportion thereof or, to a solid support so that it can be readily washedfree of unbound reactants.

α_(1d)AR, or portion thereof, suitable for use in the cell-free assaysdescribed above can be isolated from natural sources (e.g., as membranepreparations derived from bladder, e.g., human bladder) or preparedrecombinantly or chemically. The α_(1d)AR, or portion thereof, or can beprepared as a fusion protein using, for example, known recombinanttechniques. Preferred fusion proteins include a HIS tag, a FLAG tag, aGFP tag or other tag (moiety) suitable for use in colorimetric assays.Typically, the non-α_(1d)AR moiety is present in the fusion proteinN-tertninal to the α_(1d)AR, or portion thereof domain, but it can alsobe C-terminal.

As indicated above, the α_(1d)AR, or portion thereof, can be presentlinked to a solid support, including a plastic or glass plate or bead, achromatographic resin, a filter or a membrane. Methods of attachment ofproteins, or membranes containing same, to such supports are well knownin the art.

The binding assays of the invention also include cell-based assays inwhich α_(1d)AR, or portion thereof, is associated with the cell membraneof an intact cell. Cells suitable for use in such assays include cellsthat naturally express α_(1d)AR and cells that have been engineered toexpress, advantageously, over express, α_(1d)AR (or portion thereof).Advantageously, cells expressing human α_(1d)AR are used. Suitable cellsare preferably eucaryotic, including mammalian (human and nonhuman)cells, insect cells and yeast cells.

Cells can be engineered to express α_(1d)AR (advantageously, humanα_(1d)AR, or portion thereof) by introducing into a selected host (e.g.a eucaryotic host) an expression construct comprising a sequenceencoding α_(1d)AR, or portion thereof, operably linked to a promoter. Avariety of vectors and promoters can be used. (See Schwinn et al, J.Pharm. Exp. Ther. 272: 134 (1995).) Introduction of the construct intothe host can be effected using anv of a variety of standardtransfection/transformation protocols (see Molecular Biology, ALaboratorv Manual, second edition, J. Sambrook, E. F. Fritsch and T.Maniatis, Cold Spring Harbor Press, 1989). Cells thus produced can becultured using established culture techniques suitable for the involvedhost. Culture conditions can be optimized to ensure expression of theα_(1d)AR (or portion thereof) encoding sequence.

While for the cell-based binding assays it is appropriate that theα_(1d)AR (or portion thereof) be associated with the cell membrane, forother purposes the expression product can be secreted into the culturemedium or present in the cell cytoplasm.

The cell-based binding assays of the invention can be carried outessentially as described above with respect to the cell free assays.Advantageously, the cell used expresses predominantly the α_(1d)ARsubtype. By way of example, the cell-based binding assay can be carriedout by adding test compound (advantageously, bearing a detectable (e.g.,radioactive or fluorescent) label), to medium in which the α_(1d)AR (orportion thereof) expressing cells are cultured, incubating the testcompound with the cells under conditions favorable to binding and thenremoving unbound test compound and determining the amount of testcompound associated with the cells.

As in the case of the cell-free assays, the cell-based assays can alsotake the form of competitive assays, as described above. For example, acompound known to bind α_(1d)AR (and preferably labelled with adetectable label) can be incubated with α_(1d)AR (or portion thereofexpressing cells in the presence and absence of test compound. Theaffinity of a test compound for α_(1d)AR can be assessed by determiningthe amount of known compound associated with the cells incubated in thepresence of the test compound, as compared to the amount associated withthe cells in the absence of the test compound.

A test compound identified in one or more of the above-described assaysas being capable of binding to α_(1d)AR can, potentially, serve as anα_(1d)AR antagonist and therefore be suitable for use in the irritativesymptom treatment method of the invention. To determine the specificeffect of any particular test compound selected on the basis of itsability to bind α_(1d)AR, various assays can be used including IP assays(see Schwinn et al, J. Pharm. Exp. Ther. 272:134 (1995)) and bladder(e.g. human bladder) smooth muscle contraction assays (Ford et al, Mol.Pharm. 49:209 (1996)). Compounds suitable for use in treating irritativesymptoms will be associated with antagonistic (inhibitory) effects inthe IP assay and contraction inhibitory effects in the contractionassay.

In another embodiment, the invention relates to compounds identifiedusing the above-described assays as being α_(1d)AR antagonist. Thecompounds identified in accordance with the above assays can beformulated as pharmaceutical compositions. Such compositions comprisethe compound and a pharmaceutically acceptable diluent or carrier. Thecompound can be present in dosage unit form (e.g., as a tablet orcapsule) or as a solution, preferably sterile, particularly whenadministration by injection is anticipated. The dose and dosage regimenwill vary, for example, with the patient, the compound and the effectsought. Optimum doses and regimens can be determined readily by oneskilled in the art.

In another embodiment. the invention relates to antibodies specific forα_(1d)AR, and antigen binding fragments thereof, including F(ab)₂′ orF(ab) fragments. The antibodies can be monoclonal or polyclonal and canbe prepared using standard techniques. The antibodies can be used inα_(1d)AR purification protocols or the antibodies can be formulated aspharmaceutical compositions and used therapeutically as α_(1d)ARantagonists.

In yet another embodiment, the present invention relates to a genetherapy approach to treating irritative symptoms. In this embodiment,oligonucleotides (constructs) are used that, upon administration, resultin the production of a molecule that down regulates production ofα_(1d)AR. In a related embodiment, the present invention relates toα_(1d)AR antisense constructs and to a method of using same to treatirritative symptoms. Such constructs can be designed to target any of avariety of regions of the α_(1d)AR gene, including the encoding sequence(e.g., regions encoding the intracellular portion that interacts with Gprotein and participates in the signal transduction pathway) and the5′-untranslated region.

Delivery of the above-described constructs can be effected using any ofa variety of approaches, including installation into the bladder (e.g.via the uretha) and introduction into the cerebrospinal fluid. Theconstructs can also be administered systemically, in which casetargeting can be effected using, for example, smooth muscle (e.g.bladder smooth muscle) specific promoters.

Effective vectors for use in the above-described gene therapy/antisenseembodiments include viral vectors, such as retroviral vectors,adenoviral vectors and adenoassociated viral vectors. The constructs canalso be present in association with a lipid, e.g. a liposome. (Fordetails of antisense constructs and delivery systems, etc. see, forexample, Wagner Nature 372:333 (1994).) The amount of construct to beadministered will vary, for example, with the construct, the patient andthe effect sought. One skilled in the relevant art can readily optimizethe dose and treatment regimen.

In yet another embodiment, the invention relates to kits, for example,kits suitable for conducting assays described herein. Such kits caninclude α_(1d)AR, or portion thereof, for example, bound to a solidsupport. The kit can include an α_(1d)AR-encoding sequence, α_(1d)ARantisense construct or α_(1d)AR-specific antibody. The kit can includeany of the above components disposed within one or more container means.The kit can further include ancillary reagents (e.g., buffers) for usein the assays.

Certain aspects of the present invention are described in greater detailin the non-limiting Examples that follow.

EXAMPLES

The following experimental details are relevant to the specific Examplesthat follow.

Tissue preparation. Full-thickness human bladder detrusor was obtainedas discarded “normal” tissue adjacent to tumor specimens (n=1 radicalcystectomy, n=12 radical cvstoprostatectomy for transitional cellcarcinoma of the bladder) with appropriate institutional approval. Eachsample was inspected by a pathologist, and normal tissue confirmed.Detrusor smooth muscle was grossly teased from urothelial and serosallayers, snap frozen in liquid nitrogen within 30 minutes of excision,and stored at −70° C. for later use. Whole rat bladder was obtained fromeuthanized male Sprague-Dawley rats (Charles River Laboratories;Wilmington, Mass.) with institutional animal care committee approval.Rat tissue was harvested within two minutes of death, snap frozen inliquid nitrogen, and stored at −70° C. for later use.

Human detrusor and rat bladder membrane preparation. Human detrusor andrat whole bladder was minced over dry ice, and suspended in cold lysisbuffer (5 mM Tris HCl and 5 mM EDTA, pH 7.4) with protease inhibitorsbenzamirdine (10 mg/ml), leupeptin (5 mg/ml), and soybean trypsininhibitor (10 mg/ml) (Sigma Chemical Company; St. Louis, Mo.). A lysatewas prepared with a Polytron PT 3000 (Brinkmann; Westbury, N.Y.) at10,000 rpm for 10 seconds. After pelleting at 40,000×g for 15 minutes(Sorvall SM24 rotor), membranes were suspended in cold resuspensionbuffer (150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, pH 7.4) with proteaseinhibitors, and kept on ice for immediate use (or stored at −70° C. forlater use). Protein content was determined using the bicinchoninic assay(BCA) with bovine serum albumin (BSA) standards (Pierce; Rockford,Ill.).

Radioligand binding. All mRNA and protein studies described wereperformed using detrusor from each patient described above (n=13). Inorder to conserve sample, and yet fully characterize α₁ARs in humandetrusor, additional full saturation binding isotherms were generated inhuman detrusor samples from a subset of patients (n=5) using a bufferconsisting of 150 mM NaCl, 50 mM Tris-HCl and 5 mM EDTA. pH 7.4, withprotease inhibitors. Each reaction was performed in triplicate, in atotal volume of 0.25 ml, including diluted human detrusor membranes (50to 100 mg protein) and the α₁AR antagonist [¹²⁵I]HEAT (NEN ResearchProducts-DuPont; Boston, Mass.) ranging in concentration from 2-900 pM;nonspecific binding was measured in the presence of 1 mM prazosin(Sigma). The reaction proceeded at 25° C. for 45 minutes. and wasterminated with five-fold dilution of ice-cold 50 mM Tris HCl, pH 7.4buffer, followed by rapid filtration over GF/C filters using a Brandelharvester. Dried filters were then counted in a gamma counter. Specificbinding was calculated by subtracting nonspecific binding from totalbinding. Saturation curves were fit with noniterative regressionanalysis using InPlot software (GraphPad; San Diego, Calif.). Total α₁ARdensity was then determined in each detrusor sample as described above,using a saturating concentration of [¹²⁵I]HEAT (300 pM). Results arereported as mean±SEM to two significant figures.

To determine Ki values in human detrusor for α₁AR subtype discriminatingligands, competition binding was performed in triplicate in a totalvolume of 0.25 ml using binding buffer (see saturation binding above).Human detrusor membranes (50 to 100 μg protein) were incubated with aK_(d) concentration (120 pM) of the α₁AR antagonist [¹²⁵I]HEAT, andincreasing concentrations (10⁻¹² to 10⁻¹³M) of the non-radiolabeledα_(1d)AR-selective ligand BMY7378 (Research Biochemicals International;Natick, Mass.). Reaction conditions were as described above. Curves werefit with noniterative regression analysis using InPlot software(GraphPad).

Preparation of RNA. Total RNA was extracted from human detrusor or ratwhole bladder samples using the RINazol method (Tel-Test, Inc.;Friendswood, Tex.). RNA was quantitated using a spectrophotometer at260/780 nm, and aliquoted into 20 mg samples for immediate use.

Human α₁AR cDNA constructs. The human α_(1a)AR probe consists of a 0.326kb (PvuII/HindIII) fragment in pGEM-4Z (Promega Corporation; Madison,Wis.), corresponding to nucleotides 958-1283 of the cloned humanα_(1a)AR cDNA (GenBank #L31774). The human α_(1b)AR probe consists of a0.673 kb (XhoI/BamHI) fragment in pGEM-4Z (Promega), corresponding tonucleotides 94-766 of the cloned human α_(1b)AR cDNA (GenBank #L31773).The human α_(1d)AR probe consists of a 0.377 kb (EcoRI/PstI) fragment,corresponding to nucleotides 520-896 of the cloned human α_(1d)AR cDNA(GenBank #L31772). FIG. 1 shows the location of each α₁AR subtype probewithin a schematic of the encoded protein. The human cyclophilin probeconsists of a 0.103 kb (KpnI/EcoRI) fragment in pTRI (Ambion, Inc.;Austin, Tex.), corresponding to nucleotides 38-140 of the cloned humancyclophilin gene (GenBank #X52856).

Labeling of RNA probes. Antisense single-stranded radiolabeled RNAprobes were generated from linearized α₁AR cDNA constructs using RNApolymerase T7 (α_(1a), cyclophilin) and SP6 (α_(1b), α_(1d)) asdescribed in the Promega Protocols and Applications Guide (PromegaCorporation; Madison, Wis.). α_(1a)AR and α_(1d)AR cDNA constructs werelinearized with EcoRI, and the α_(1b)AR cDNA construct was linearizedwith HindIII. ³²P-αUTP (NEN Research Products-DuPont) was incorporatedinto RNA probes at the time of probe synthesis. All probes were purifiedon a 5% polyacrylaminde gel (300V for 1.5 hr); after exposure to filmfor 3 min, radiolabeled RNA probes were excised from the gel andpassively eluted overnight into 400 μl of RPA II kit (Ambion) elutionbuffer at 37° C.

RNase protection assays. RNase protection assays were conducted aspreviously described (Zinn et al, Cell 34:865-879 (1983)) with a fewmodifications. In brief, total RNA samples (20 mg) were dissolved in 20ml of hybridization buffer containing >20-fold excess of radiolabeledprobe (2×10⁵ cprn/reaction for α_(1a), α_(1b), α_(1d), and 1×10⁵cpm/reaction for cyclophilin), and incubated overnight at 55° C.(α_(1a), α_(1b)) and 65° C. (α_(1d), cyclophilin). To ensure specificityof the synthesized radiolabeled antisense human α₁AR subtype selectiveprobes, RNase protection assays were performed in tandom with total RNAextracted from rat-1 fibroblast cells stably expressing each clonedhuman α₁AR subtype. As a negative control, RNase protection assays foreach α₁AR subtype selective probe were performed in tandom with yeasttRNA samples and other non-hybridizing α₁AR subtypes. Antisenseradiolabeled probe to the highly conserved region of the constitutivelyexpressed human cyclophilin gene was also utilized as a control toensure identical amounts of total RNA in each assay. The final gel wasexposed to X-Omat AR film (Eastman Kodak Company; Rochester, N.Y.) for24-72 hours.

α₁AR mRNA quantitation in human detrusor smooth muscle from RNaseprotection assays. In order to quantitate relative α₁AR subtype mRNA,each RNase protection assay final gel was exposed to PhosphorImagerplates (Molecular Dynamics; Sunnyvale, Calif.) for 24 hours. Volumeintegration of specific protected radiolabeled bands for each mRNAresulting from hybridization products was corrected for background,normalized for cyclophilin signal, and expressed as arbitrary densitvunits, using ImageQuant gel image-analysis software (MolecularDynamics). α₁AR probes contained the following number of UTP sites for³²P-αUTP incorporation: α_(1a) 88, α_(1b) 117, α_(1d) 51. Arbitrarydensity units were normalized to the lowest ³²P-αUTP incorporating probe(α_(1d)AR) and then expressed as a fraction (±SEM) of total α₁AR mRNAsignal strength.

Polymerase Chain Reaction (PCR). RT-PCR was used to confirm expressionof human detrusor α₁AR subtypes (to ensure low concentrations of asubtype were not missed in human bladder) and to compare α₁AR subtypemRNA expression in rat whole bladder with previously published rat data(Walden et al, J. Urol. 157:1032-1038 (1997), Scofield et al, J.Pharmacol. Exper. Ther. 275:1035-1042(1995)). Human and rat α₁AR subtypeprimers were synthesized at Duke University Medical Center. Reversetranscription of 1 mg of DNase-treated human detrusor or rat bladder RNAwas performed in triplicate in a 20 ml reaction mixture containing 5 mMMgCl₂, 1 mM each of dATP, dCTP, dGTP, and dTTP, 10 mM TrisHCl, 50 mMKCl, 2 ml DEPC treated water, 2.5 mM random hexamers, 1 unit of RNaseinhibitor, and 2.5 units of MuLV Reverse Transcriptase (Perkin Elmer;Foster City, Calif.); simultaneous control samples not treated withreverse transcriptase were used to rule out amplification of genomicDNA. Reverse transcriptase reactions were run for 60 min at 42° C. 5 minat 95° C., and 10 min at 4° C. Each α₁AR mRNA subtype was amplified byPCR in triplicate in a 100 ml reaction containing 50 mM KCl, 10 mMTris-HCl, pH 8.3, 2 mM MgCl₂, 200 mM each of DATP, dCTP, dGTP and dTTP,15 pM of sense and antisense primer. 5% DMSO, and 2.5 units of AmpliTaqDNA polymerase (Perkin Elmer). PCR reactions were performed in aDeltaCycler II™ temperature cycler (ERICOMP; San Diego, Calif.). Thefollowing conditions were established for all three rat primer sets: onedenaturation cycle for 3 minutes at 95° C., 35 cycles of 1 min at 95°C., 1 min annealing at 58° C. and a 1 min extension at 72° C. Thefollowing conditions were established for all three human primer sets:one denaturation cycle for 3 minutes at 95° C., 35 cycles of 1 min at95° C. 1 min annealing at 60° C. for α_(1a) and α_(1b), and 68° C. forα_(1d), and a 1 min extension at 72° C. A final extension cycle wasperformed for 10 min at 72° C. Reaction mixtures were then cooled at 4°C. 10 ml of each PCR product was separated bv gel electrophoresis in0.8% agarose. Since PCR experiments were only confirmatory in nature bydesign, exact quantitation (requiring competitive PCR) was notperformed. However, to ensure that any statement regarding relative mRNAlevels is appropriate, it is important to note that conditions describedabove (e.g. different annealing temperatures) were chosen afterextensive preliminary analysis with each primer set to ensure optimalamplification conditions with similar primer product efficiency.Equality of reverse transcription efficiency for products was checkedusing equal concentrations of starting control cDNA; these reactionsalso served as a positive control for use of correct primer sets.Thirty-five cycles of amplification was chosen since it is at the upperend of the linear amplification range for all six primer sets (α₁ARmRNAs are rare at baseline in many human tissues and in our hands 40cycles of amplification is where the curve becomes non-linear).

Example 1 Human Patient Population

Human detrusor smooth muscle was obtained from male (n=12) and female(n=1) patients. Patient age ranged from 56 to 76 years old (mean=59.6).Significant past medical history included tobacco abuse, coronary artervdisease, hypertension controlled with α₁AR or PAR antagonists (n=3), anda history of BOO (n=2) necessitating previous transurethral resection ofprostate. Comparison of results from patients with hypertension and/orBOO (n=5) suggests medical history did not affect the results. A largerstudv would be required to make any definitive statement in this regard.

Example 2 α₁AR Ligand Saturation Binding

Pharmacological characteristics of α₁ARs in human detrusor include a Kdfor the radiolabeled α₁AR antagonist [¹²⁵I]HEAT of 130±1.9 pM, similarto that reported for cells stably expressing the cloned human α_(1a)ARsubtype ((Schwinn et al, J Pharmacol. Exper. Ther. 272:134-142 (1995)).A representative saturation binding isotherm is shown in FIG. 2. Totalα₁AR density as measured by saturation binding in human detrusormembrane preparations with the α₁AR antagonist [¹²⁵I]HEAT is 6.3±1.0fmol/mg protein (mean±SEM, range 2.7-9.0, n=13). Although low (withcorresponding high non-specific binding of 70-80% as expected), α₁ARexpression is reproducible and consistent within and between patients.

Example 3 Identification and Quantification of the α₁AR mRNA Subtypes inHuman Bladder Detrusor

In order to determine which α₁AR subtypes are present in human detrusor,molecular approaches were chosen due to their sensitivity andspecificity. To ensure specificity of the synthesized radiolabeledantisense human α₁AR subtype selective probes, RNase protection assayswere performed simultaneously with total RNA extracted from rat-1fibroblast cells stably expressing each cloned human α₁AR subtype. Eachα₁AR subtype specific probe protects a single predominant fragment ofpredicted size without cross-hybridization (FIG. 3, positive controlcells); a lack of cross-hybridization between subtypes with each probe(Price et al, Mol. Pharmacol. 46:221-226 (1994)). As was previouslydemonstrated a further negative control, RNase protection assays foreach α₁AR subtype selective probe were performed in tandom with yeasttRNA samples, where no hybridization is demonstrated (FIG. 3, tRNAlane). Human detrusor contains α_(1d)AR>α_(1a)AR mRNA, but no α_(1b)ARmRNA in every patient studied (n=13; FIG. 3 shows representative resultsfrom patients number 1 through 5). This data, when corrected forbackground, normalized for cyclophilin content, and corrected for probe³²P-αUTP incorporation, reveals that α_(1d)AR mRNA constitutes 66±4.8%and α_(1a)AR mRNA 34±4.8% of the total α₁AR mRNA in human detrusor.

Example 4 Confirmation of α₁AR Subtype mRNA in Human Detrusor andComparison with Rat Whole Bladder using RT-PCR

In order to confirm results from RNase protection assays and to comparewith another frequently used animal model (rat), α₁AR subtype expressionwas examined using RT-PCR in each patient. Primer nucleotide sequences,melting temperatures (T_(m)), and primer positions relative to the cDNAsequence are shown in Table 1 and Table 2; these primers do not span anintron. TABLE 1 Oligonucleotide primers used for rat α₁AR subtypeRT-PCR. primer position Rat α₁AR nucleotide sequences relative primers5′ 3′ T_(m) to cDNA α_(1a)AR sense GTAGCCAAGAGAAAGCCG 62° C. 628-647α_(1a)AR CAACCCACCACGATGCCCAG 66° C. 839-820 antisense α_(1b)AR senseGCTCCTTCTACATCCCGCTCG 68° C. 629-649 α_(1b)AR AGGGGAGCCAACATAAGATGA 62°C. 928-908 antisense α_(1d)AR sense CGTGTGCTCCTTCTACCTACC 66° C. 759-779α_(1d)AR GCACAGGACGAAGACACCCAC 68° C. 1062-1042 antisenseThe nucleotide sequences listed above correspond to the followingsequence identifiers, respectively: SEQ ID NOs: 1-6.

TABLE 2 Oligonucleotide primers used for human α₁AR subtype RT-PCR.primer position Human α₁AR nucleotide sequences relative primers 5′ 3′T_(m) to cDNA α_(1a)AR sense ATCATCTCCATCGACCGCTACA 66° C. 355-376α_(1a)AR TCACTTGCTCCGAGTCCGACTT 68° C. 697-676 antisense α_(1b)AR senseGCTCCTTCTACATCCCTCTGG 68° C. 629-649 α_(1b)AR AGGGTAGCCAGCACAAGATGA 67°C. 928-908 antisense α_(1d)AR sense ACCACGCGCAGCCTCGAGGCAGGC 84° C.850-873 α_(1d)AR GAGCGAGCTGCGGAAGGTGTGGCC 82° C. 999-976 antisenseThe nucleotide sequences listed above correspond to the followingsequence identifiers, respectively: SEQ ID NOs: 7-12.

Although RNase protection assays are considered the “gold standard” forquantitating mRNA present in a given tissue, this approach is not assensitive as PCR, therefore very small amounts of mRNA can be missed ina RNase protection assay but demonstrated by PCR. As shown in FIG. 4,RT-PCR performed on human detrusor total RNA demonstrates the presenceof α_(1a)AR and α_(1d)AR mRNA, and lack of α_(1b)AR mRNA, consistentwith data from the RNase protection assays. Of note, α_(1d)AR mRNAaccounts for approximately 60-70% of total α₁AR mRNA in human detrusorwith α_(1a)AR mRNA accounting for 30-40%. again confirming the RNaseprotection assay results. Species heterogeneity (human versus rat) ofα₁AR subtype rnRNA expression has been previously reported for manytissues (Price et al, Mol. Pharmacol. 46:221-226 (1994), Price et al,Mol. Pharmacol. 45:171-175 (1994)). Indeed, as seen in FIG. 4, RT-PCRperformed on pooled rat bladder total RNA demonstrates the presence ofall three α₁AR mRNAs in roughly equal concentrations in rat.

Example 5 Determination of α₁AR Subtype Expression at a Protein Level

Competition analysis was used to determine α₁AR subtype expression at aprotein level in human detrusor. Since molecular studies demonstrate apredominance of α_(1d)AR mRNA, the α_(1d)AR-selective compound BMY7378was used in these studies. As graphically represented in FIG. 5, atwo-site fit was evident in every patient studied (n=13), with highaffinity binding predominating (high pK_(i)=8.6±0.2 [66±3.1% total] vs.low pK_(i)=4.9±0.2 [35±3.1% total]) (Table 3). TABLE 3 Results fromcompetition binding experiments utilizing membranes from rat-1fibroblasts stably transfected with each α₁AR subtype (controls) andhuman detrusor (n = 13). Since no α_(1b)AR was found in human detrusorby RNase protection assays and RT-PCR, one versus two site fit of thedata was utilized. BMY7378 (pK_(i)) α_(1a)AR α_(1b)AR α_(1d)AR % High %Low Human detrusor 4.9 ± 0.2 — 8.6 ± 0.2 66 ± 3.1 35 ± 3.1 Controlα_(1a) 4.8 ± 0.1 — — Control α_(1b) — 5.1 ± 0.3 — Control α_(1d) — — 8.5± 0.1

All documents cited above are hereby incorporated in their entirety byreference.

One skilled in the art will appreciate from a reading of this disclosurethat various changes in form and detail can be made without departingfrom the true scope of the invention.

1-10. (canceled)
 11. A method of screening a test compound for itsability to bind α_(1d)AR comprising incubating said test compound withα_(1d)AR, or portion thereof, and determining the amount of said testcompound bound to said α_(1d)AR, or portion thereof.
 12. The methodaccording to claim 11 wherein said portion comprises the transmembraneportion of α_(1d)AR.
 13. The method according to claim 11 wherein saidtest compound bears a detectable label.
 14. The method according toclaim 11 wherein said α_(1d)AR, or portion thereof, is present in a cellmembrane.
 15. The method according to claim 14 wherein said membrane isthe membrane of an intact cell.
 16. The method according to claim 15wherein said cell is a eucaryotic cell.
 17. The method according toclaim 15 wherein said cell is a cell that has been engineered to expressor over-express said α_(1d)AR, or portion thereof.
 18. The methodaccording to claim 11 wherein said test compound is incubated with saidα_(1d)AR, or portion thereof, in the presence of an agent known to bindto α_(1d)AR, or portion thereof, and the amount of said test compoundthat binds to said α_(1d)AR, or portion thereof, is determinedindirectly by determining the amount of said agent that binds to saidα_(1d)AR, or portion thereof.
 19. The method according to claim 18wherein said agent bears a detectable label.
 20. The method according toclaim 18 wherein said agent is BMY7378 or tamsulosin. 21-27. (canceled)28. A method of identifying a candidate agent suitable for use inrelieving irritative symptoms of bladder or lower urinary tract diseasein a patient comprising incubating a test compound with α_(1d)AR, orportion thereof, and determining the amount of said test compound boundto said α_(1d)AR, or portion thereof, wherein a test compound that bindssaid α_(1d)AR, or portion thereof, is said candidate agent.
 29. Themethod according to claim 28 wherein said portion comprises thetransmembrane portion of α_(1d)AR.